DESCRIPTION: Critical limb ischemia (CLI) in patients is characterized by ulceration, pain, and limb loss with associated high mortality. For CLI patients who are not suitable surgical candidates, stem cell therapy represents a promising alternative to increase perfusion. However, results from recent clinical trials with bone marrow-derived stem cells indicate a lack of lasting efficacy due to two major obstacles: poor long-term tissue engraftment and their low potency. This grant addresses these obstacles by parallel development of 1) thermosensitive injectable polymers that enable improved survival and proliferation of stem cells in ischemic tissue 2) efficient generation of MSCs which have been partially preprimed towards the endothelial lineage and exhibit higher potency. To accomplish these goals, a mouse MSC line genetically tagged with a lacZ reporter driven by an endothelial promoter (flk1) was generated to perform high-throughput testing of multiple vascular differentiation protocols. This screen identified laminin I and collagen IV as potent molecular inducers of endothelial differentiation of mouse and human MSCs. Endothelial prepriming of MSCs dramatically improved ischemic perfusion in mice. The hypothesis to be tested is that preprimed MSCs with high reparative potency injected with thermosensitive polymers which gel at body temperature will target stem cells to ischemic foci to promote their engraftment and therapeutic potency. The project seeks to understand the molecular factors and signaling pathways involved in stepwise endothelial differentiation of MSCs and to generate a combinatorial therapy using bioengineered injectable polymer to deliver the preprimed human MSCs with peptide-based therapeutics to improve perfusion in a mouse model of hindlimb ischemia. The long-term goal is to develop a non-invasive stem-cell therapy to augment limb perfusion in patients with CLI.